Applications
Regulators of Apoptosis
- Bcl-2, Bax, Bcl-XL
- Fas
- XIAP, Survivin
- TUNEL, Mitochondrial Membrane Potential
- Caspase Cleavage
- Phosphatydil Serine (PS)/Annexin V
- DNA Fragmentation
Novel Progenitor/Stem Cell Candidates
- CD34+ 38- Lin-
- CD34- Lin- “SP”
- Normal (NOD/scid)
- AML (NOD/scid) - CD34+ KDR+ (VEGF-R2)
Flow Cytometry Applications
Apoptosis
Apoptosis Marker
- Propidium Iodide (PI)
- Annexin V
- TUNEL (TdT)
- CMXRos
- Mitochondrial Mass
- Caspase
- Acridine Orange (AO)
Feature Measured by FCM
- Apoptosis/Necrosis
- Early Apoptosis
- DNA Strand Breaks/Apoptosis
- Apoptosis/Mitochondrial Membrane Potential Mitotracker
- Apoptosis-Pathway specific
- Apoptosis/Necrosis
The correct combination of two, three or four of these markers can reveal specific features of stage or extent of apoptosis. Selection of markers depends on the hypothesis to be experimentally tested.
Figure 1: Population in green represents Annexin V positive cells, those in red represent PI positive and populatino in purple represents cells positive for both Annexin V and PI.
Figure 2: Caspase activation (middle panel), PS/Annexin V expression (lower panel) and changes in mitochondrial membrane potential can be directly correlated with each other in this assay.
Cell Cycle/Ploidy Analysis
These dyes discriminate between different phases of cell cycle. Choosing a particular dye and protocol depends on the cell sample type and on whether the staining/analysis can be performed on fixed cells.
Dye Name
- DAPI
- Acridine Orange (AO)
- Propidium Iodide (PI)
- Hoechst 33342
- BromoDeoxyuridine
Feature Measured by FCM
- DNA Content
- DNA/RNA discrimination
- DNA Content
- DNA Content
- DNA Replication (S-Phase)
Fixed/Viable
- Fixed
- Viable
- Viable
- Viable
- Fixed
Ki67 discriminates between G0 and G1 cells, PI between G1, S-phase and G2M.
Multifluorochrome Immunophenotyping
Immunophenotyping characterizes subsets of cells according to the proteins or receptors expressed on the cell surface. The example below uses size, in the upper left graph, along with CD3-FITC, CD45-PerCP and Sca-1-PE to enumerate stem cell populations in mouse bone marrow.
Cell Sorting
The FACS Vantage instruments have the capability of selectively removing specific subsets from heterogenous cell suspensions.
Sort purity routinely exceeds 98%. Cells can be deposited automatically into microtiter wells (any number selected).
Laser Scanning Cytometry
The Laser Scanning Cytometer allows the quantitation of fluorescence intensity of individual cells deposited on slides. This technology for localization studies and quantitation of cellular components with the capacity of population analysis and imaging. These are examples of protein quantitation using the LSC system (courtesy of Dr. Stevan Kornblau).
Confocal Microscopy
Co-localization of Bcl-2 and Calreticulin in PC-3 cells
Bovine cardiac muscle cell